A micro particle analyzing device according to the prior art, for example, a flow cytometry analyzing a blood cell such as erythrocyte or leukocyte in blood uses a flow cell having a rectangular cross section and makes a sample solution, which includes micro particles to be analyzed, flow to a central part thereof. FIG. 1 shows an optical part for analyzing the sample flowing in the flow cell having the rectangular cross section. This structure has a characteristic of transverse illumination. That is, an incident path of a laser light source intersects an emission path of fluorescence generated by a micro particle excited due to the illumination from the light source.
The transverse illumination device for analyzing micro particles comprises a light source section 10 emitting light which will be illuminated to a sample; a cylindrical lens 20 regulating an amount and a focal distance of the light emitted from the light source 10; an object lens 40 adjacent to the sample to condense light emitted from the sample; a reflecting mirror 50 for reflecting the light having passed through the object lens to a reading section 80; and a reading section 80 sensing and reading the light emitted from the sample.
To analyze the erythrocyte or leukocyte in fluid, fluid is made to flow in a flow cell 90 mounted to the micro particle analyzing device.
FIG. 2 shows a cross section of the flow cell. The fluid flows to a sample injecting inlet 91, and a buffer solution 94 is injected to a buffer solution injecting inlet 92 to flow around the sample. The sample responds to light of the light source 10 and emits light when passing through an observation point 93.
Preferably, the flow cell 90 or the sample is treated with fluorescent material in advance. When the micro particle (for example, erythrocyte) in the sample is illuminated with light of the light source 10, it responds the light together with the fluorescent material and emits light having a specific wavelength band. The light is selectively passed through a filter 70 and then can be signal-processed at a signal processing reading section 80 (for example, photo multiplier tube (PMT) or CCD camera).
The above micro particle analyzing device may further comprise an aperture 60 and the filter 70 so that only light having a specific wavelength band of the emission lights can be passed through.
In recent years, a microchip based flow cytometry using a microchip including a micro channel as a flow cell to analyze a micro amount of sample has been researched and developed. When using a microchip 540 as shown in FIG. 6, since the microchip has a flat horizontal structure, it is adopted an optical structure such that laser light is incident through an object lens and fluorescence emitted from a micro particle is focused at the same time (i.e., axial illumination).
The transverse illumination shown in FIG. 1 can mount different standards of optical devices at the incident path of the light source and the emission path of the fluorescence. However, in the axial illumination as shown in FIGS. 3 and 4, characteristics of the incident light and the emission fluorescence are determined depending on the standard of the object lens.
The above characteristic of the axial illumination causes a disadvantage that it is impossible to independently regulate a light spot form of an incident light and a focused form of emission fluorescence each other.
To secure a uniformity of scattered light or fluorescence signal emitted from a sample particle in the microchip based flow cytometry, a magnitude of the light spot of the incident laser light should be large enough to include the sample particle. In addition, it should emit regular lights without regard to upper and lower positions of the particles to the light spot. Accordingly, it is preferred to use a lens having a small numerical aperture. However, when the emission light of the micro particle is again focused with the lens having the small numerical aperture, there is a problem of decreasing a focusing efficiency of emission light. The reason is because a lens having a large numerical aperture is preferred to focus the emission light of the micro particle.
Accordingly, ideal conditions of the optical part applied to the flow cytometry are as follows. An incident light is made to pass through a lens having a small numerical aperture and then to illuminate the sample particle. An emission light is made to pass through a lens having a large numerical aperture and then to be detected at a sensing section.